The overall objective of the proposed research is to characterize the structure and function of dark basal keratinocytes in normal as well in carcinogen and/or promoter treated skin. Dark cells will be studied by light and electron microscopy using plastic embedded material. The percentage of dark cells in the basal layer, the thymidine labeling index of the basal layer and of the dark cell population, the volume densities of tonofilaments, ribosomes-conglomerates, and the number of desmosomes and hemidesmosomes will be determined using morphometric techniques in normal adult and fetal epidermis as well as in epidermis treated with DMBA as initiator and TPA as promoter. The sequential changes taking place in the dark cell population will be analyzed at various time points during this two stage carcinogenesis protocol. The influence of hair follicles in the overall production of dark cells will be studied by quantitatively comparing the dark cell population of hairy skin with that of globrous skin and keratinized oral mucosa in both normal and TPA epithelia. The efficiency of various weak and strong promoters as well as non-specific stimuli such as skin abrasion, to induce dark cells will be determined in order to verify the partially proven hypothesis that increased induction of dark cells is a reliable marker of tumor promotion efficiency. Isolation of dark cells from normal and TPA treated epidermis will be attempted using differences in detachment velocities during EDTA incubation and/or gradient centrifugation. Isolated dark cell fractions as well as tissue sections will be used to detect specific biological markers. Gamma glutamyltransferase will be monitored histochemically and biochemically in dark cells from normal, TPA treated, and DMBA + TPA treated epidermis in order to assess the validity of its use as a marker of epithelial dedifferentiation. Other possible markers such as carcinoembryonic antigen and fetal keratins will be studied in the same material by histoimmunochemistry.